EP3407916B1 - Lieurs de phosphonate et leur utilisation pour faciliter la rétention cellulaire de composés - Google Patents

Lieurs de phosphonate et leur utilisation pour faciliter la rétention cellulaire de composés Download PDF

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EP3407916B1
EP3407916B1 EP17744740.6A EP17744740A EP3407916B1 EP 3407916 B1 EP3407916 B1 EP 3407916B1 EP 17744740 A EP17744740 A EP 17744740A EP 3407916 B1 EP3407916 B1 EP 3407916B1
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antibody
group
seq
moiety
compound
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EP3407916A2 (fr
EP3407916A4 (fr
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Robert M. Garbaccio
Jeffrey C. KERN
James J. MULHEARN
Philip E. Brandish
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Merck Sharp and Dohme LLC
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Merck Sharp and Dohme LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/3804—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se) not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
    • C07F9/40—Esters thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to phosphonate linkers which are suitable to facilitate intracellular retention of compounds with passive cell permeability.
  • Antibody drug conjugates are targeted chemotherapeutic molecules combining the ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, internalizing the ADC, and releasing the drug from the ADC, thereby enhancing the drug's anti-tumor activity.
  • This strategy has met limited success in part because many cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • Promising advancements with immunoconjugates has seen cytotoxic drugs linked to antibodies through a linker that is cleaved at the tumor site or inside tumor cells.
  • the successful ADC development for a given target antigen depends on optimization of antibody selection, linker design and stability, drug potency and mode of drug and linker conjugation to the antibody.
  • Linker properties of pH and redox sensitivities and protease susceptibility influence circulatory stability and release of the drug moiety.
  • the intracellular cleavage of disulfide containing linkers of an ADC is limited by the oxidizing potential of endosomes and lysosomes and are probably not released by reductive cleavage within the endocytic pathway ( Austin et al., Proc. Natl. Acad. Sci. USA 102: 17987-17992 (2005 )).
  • Reductive cleavage may occur at the cell membrane and impart a bystander killing effect of tumor and susceptible normal cells by free drug. Inappropriate release of drug likely contributes to toxicity.
  • ADC efficacy is dependent on proteolytic cleavage for drug activity.
  • Linker stability plays an important role in both the efficacy and toxicity of ADC ( Alley et al., Bioconjugate Chem. 19:759-765 (2008 )).
  • Stable linkers such as mcc are more efficacious and safer than unstable, disulfide linkers, widening the therapeutic window. However, while mcc linkers are more stable than disulfides, they can only be used for drugs that can tolerate residual linker on it and still be potent. Thus, self-immolative linkers are needed for drugs that do not have this flexible structure activity relationship (SAR).
  • a chemical solution to targeted delivery of cytotoxic or cytostatic drugs conjugated to cell-specific ligands is the "self-immolative linker", PABC or PAB (para-aminobenzyloxycarbonyl) linker, attaching the drug moiety to the ligand in the conjugate ( Carl et al., J. Med. Chem. 24: 479-480 (1981 ); Chakravarty et al., J. Med. Chem. 26: 638-644 (1983 )).
  • PAB linker unit is also referred to as an electronic cascade spacer.
  • the amide bond linking the carboxy terminus of a peptide unit and the para-aminobenzyl of PAB may be a substrate and cleavable by certain proteases.
  • the aromatic amine becomes electron-donating and initiates an electronic cascade that leads to the expulsion of the leaving group, which releases the free drug after elimination of carbon dioxide ( de Groot, et al. Journal of Organic Chemistry 66: 8815-8830 (2001 )).
  • Cathepsin B is a ubiquitous cysteine protease with increasing activity within low pH environments (i.e. lysosomes).
  • Linkers containing the para-aminobenzyloxycarbonyl (PAB or PABC) unit, in conjunction with a peptide unit, have been developed with a "self-immolating" or “self-immolative” mechanism of 1,6 elimination and fragmentation under enzymatic, hydrolytic, or other metabolic conditions to release a drug moiety from a targeting ligand, such as an antibody ( U.S. Pat. No. 6,214,345 ; 6,677,435 5,621,002 ; 6,218,519 ; 6,835,807 ; 6,268,488 ; and 6,759,509 ; US Pat. Pub. Nos.
  • a targeting ligand such as an antibody
  • PAB type self-immolating linkers are the propensity to cause poor solubility and aggregation of the conjugates.
  • some PAB-containing conjugates may not be suitable substrates for certain cleaving enzymes or cleave too slowly to achieve efficacy.
  • PAB/PABC linkers have been exemplified for amine-terminus payloads that form stable carbamate bonds, for payloads that do not contain a linkable amine, the carbonate that is formed may not be stable and so there is a need for self-immolative linkers that can handle payloads with an oxygen terminus, for example, dexamethasone.
  • WO2015153401 discloses phosphate-based linkers comprising a payload linked to the oxygen atom of a phosphate or phosphonate group and a linker arm comprising a tuning element, an optional spacer element, and a reactive functional group capable of conjugating to a reactive group of a targeting ligand.
  • These phosphate-based linkers have a differentiated and tunable stability in blood vs. an intracellular environment (e.g. lysosomal compartment). Intracellularly, the phosphate or phosphonate group is cleaved to release the payload from the phosphate or phosphonate group.
  • phosphonate-based linkers comprising a monophosphonate, diphosphonate, triphosphonate, or tetraphosphonate group
  • a conjugate When the phosphonate-based linker is conjugated to a ligand or targeting moiety, a conjugate is provided which may be administered to an individual subcutaneously or intravenously.
  • the conjugate When the conjugate is administered to an individual, the phosphonate group is stable. However, when the conjugate is subsequently internalized or taken up by a cell, the phosphonate group is labile and is cleaved, the cleavage rate being dependent on the structure of the tuning element comprising the first linker arm.
  • a monophosphonate group remains linked to the second linker arm linked to the payload. The monophosphonate group renders the payload polar, which facilitates intracellular retention of the payload.
  • the present invention provides a payload-phosphonate-based linker compound comprising formula (I) wherein V is selected from O and S; W is a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, -S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic,
  • the compound may further comprise a cell-specific targeting ligand (L); wherein the compound comprises a compound having formula (II) wherein the reactive functional group (Z) is conjugated to an S, NR, or O group on the cell-specific targeting ligand (L).
  • the reactive functional group at the proximal end of the payload-phosphoate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the therapeutic agent.
  • the therapeutic agent may be any molecule that alters, inhibits, activates, or otherwise affects a biological event.
  • a therapeutic agent include but are not limited to, cytotoxic agent, an anti-inflammatory agent, peptide, a nucleic acid or nucleic acid analog, a small molecule, and a biomolecule.
  • the anti-inflammatory agent is a glucocorticoid receptor agonist.
  • the anti-inflammatory agent is a glucocorticoid, for example, Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, budesonide, dexamethasone, fluticasone, or mometasone.
  • the targeting ligand is an antibody or monoclonal antibody (e.g., chimeric, humanized, or human), ligand for a receptor, lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • antibody or monoclonal antibody e.g., chimeric, humanized, or human
  • ligand for a receptor e.g., lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • the chimeric, humanized, or human antibody or monoclonal antibody is an anti-Her2 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD80 antibody, anti-CD163 antibody, anti-Mucl8 antibody, anti-integrin antibody, anti-PSMA antibody, anti-CEA antibody, anti-CDl Ia antibody, anti-CTLA4 antibody, or anti-BLys antibody.
  • composition comprising a compound having formula (II) wherein V is selected from O and S; W is a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, -S(O)-, -S(O)2-, -N(R)SO2-, SO2N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl,
  • the therapeutic agent may be cytotoxic agent, an anti-inflammatory agent, peptide, or nucleic acid or nucleic acid analog.
  • the anti-inflammatory agent is a glucocorticoid receptor agonist.
  • the anti-inflammatory agent is a glucocorticoid, for example, Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, budesonide, dexamethasone, fluticasone, or mometasone.
  • the targeting ligand is an antibody or monoclonal antibody (e.g., chimeric, humanized, or human) or fragment thereof, ligand for a receptor, lectin; saccharide, poly(ethylene glycol); polysaccharide, or polyamino acid.
  • antibody or monoclonal antibody e.g., chimeric, humanized, or human
  • fragment thereof ligand for a receptor, lectin; saccharide, poly(ethylene glycol); polysaccharide, or polyamino acid.
  • the chimeric, humanized, or human antibody or monoclonal antibody is an anti-Her2 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD80 antibody, anti-CD163 antibody, anti-Mucl8 antibody, anti-integrin antibody, anti-PSMA antibody, anti-CEA antibody, anti-CDl Ia antibody, anti-CTLA4 antibody, or anti-BLys antibody.
  • the present invention further provides a compound as defined in the claims for use in the treatment of a disease or disorder.
  • the disease or disorder is an inflammatory disease or cancer.
  • the present invention further provides an anti-inflammatory compound comprising the formula as defined above, wherein D is an anti-inflammatory agent.
  • the anti-inflammatory agent is a glucocorticoid receptor agonist.
  • the anti-inflammatory agent is Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, budesonide, dexamethasone, fluticasone, or mometasone.
  • the targeting ligand is an antibody or monoclonal antibody (e.g., chimeric, humanized, or human), ligand for a receptor, lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • antibody or monoclonal antibody e.g., chimeric, humanized, or human
  • ligand for a receptor e.g., lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • the targeting ligand is a chimeric, humanized, or human anti-Her2 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD80 antibody, anti-CD163 antibody, anti-Mucl8 antibody, anti-integrin antibody, anti-PSMA antibody, anti-CEA antibody, anti-CDl Ia antibody, anti-CTLA4 antibody, or anti-BLys antibody.
  • the present invention further provides an anti-cancer compound comprising the formula as defined above, wherein D is a cytotoxic agent.
  • cytotoxic agent is selected from duocarmycins and CC-1065;
  • the cytotoxic agent is selected from a CBI (1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one)-based analogues, MCBI (7-methoxy- 1,2,9,9a- tetrahydrocyclopropa[c]benz[e]indol-4-one)-based analogues and CCBI (7-cyano- 1,2,9,9a- tetrahydrocyclo-propa[c]benz[e]-indol-4-one)-based analogue of the duocarmycins and CC- 1065.
  • CBI 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one
  • the cytotoxic agent is selected from doxorubicin and doxorubicin conjugates such as morpholino-doxorubicin and cyanomorpholino-doxorubicin.
  • the cytotoxic agent is selected from dolastatins such as dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogs, DM-I, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvaleric acid- AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloro methotrexate, 5-fluorouracil, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leuroside
  • the targeting ligand is an antibody or monoclonal antibody (e.g., chimeric, humanized, or human), ligand for a receptor, lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • antibody or monoclonal antibody e.g., chimeric, humanized, or human
  • ligand for a receptor e.g., lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.
  • the targeting ligand is a chimeric, humanized, or human anti-Her2 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD80 antibody, anti-CD163 antibody, anti-Mucl8 antibody, anti-integrin antibody, anti-PSMA antibody, anti-CEA antibody, anti-CDl Ia antibody, anti-CTLA4 antibody, or anti-BLys antibody. Also disclosed but not claimed is a method for making an antibody-drug conjugate that has reduced propensity for forming aggregates comprising:
  • composition of antibody-drug conjugates in which the propensity of the antibody-drug conjugates in the composition to form aggregates is reduced comprising:
  • acyl groups include aldehydes (-CHO), carboxylic acids (-CO 2 H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas.
  • Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyl
  • Aliphatic - As used herein, the term "aliphatic” or “aliphatic group” denotes an optionally substituted hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic ("carbocyclic") and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-12 carbon atoms. In some embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • alkenyl denotes an optionally substituted monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • the alkenyl group employed in the invention contains 2-6 carbon atoms.
  • the alkenyl group employed in the invention contains 2-5 carbon atoms.
  • the alkenyl group employed in the invention contains 2-4 carbon atoms.
  • the alkenyl group employed contains 2-3 carbon atoms.
  • Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
  • alkyl refers to optionally substituted saturated, straight or branched-chain hydrocarbon radicals derived from an aliphatic moiety containing between 1-6 carbon atoms by removal of a single hydrogen atom.
  • the alkyl group employed in the invention contains 1-5 carbon atoms.
  • the alkyl group employed contains 1-4 carbon atoms.
  • the alkyl group contains 1-3 carbon atoms.
  • the alkyl group contains 1-2 carbons.
  • alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.
  • alkynyl refers to an optionally substituted monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom.
  • the alkynyl group employed in the invention contains 2-6 carbon atoms.
  • the alkynyl group employed in the invention contains 2-5 carbon atoms.
  • the alkynyl group employed in the invention contains 2-4 carbon atoms.
  • the alkynyl group employed contains 2-3 carbon atoms.
  • Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to an optionally substituted monocyclic and bicyclic ring systems having a total of five to 10 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • aryl may be used interchangeably with the term “aryl ring”.
  • “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
  • arylalkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • Bivalent hydrocarbon chain - is a polymethylene group, i.e., -(CH 2 ) z -, wherein z is a positive integer from 1 to 30, from 1 to 20, from 1 to 12, from 1 to 8, from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 30, from 2 to 20, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or from 2 to 3.
  • a substituted bivalent hydrocarbon chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • Non-limiting examples of carbonyl groups include aldehydes, ketones, carboxylic acids, ester, amide, enones, acyl halides, anhydrides, ureas, carbamates, carbonates, thioesters, lactones, lactams, hydroxamates, isocyanates, and chloroformates.
  • Cycloaliphatic As used herein, the terms “cycloaliphatic”, “carbocycle”, or “carbocyclic”, used alone or as part of a larger moiety, refer to an optionally substituted saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring systems, as described herein, having from 3 to 10 members.
  • Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl.
  • the cycloalkyl has 3-6 carbons.
  • Halogen - As used herein, the terms “halo” and “halogen” refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I).
  • heteroaliphatic or “heteroaliphatic group” denote an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic.
  • heteroaliphatic groups contain 1-6 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur.
  • heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.
  • heteroaryl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • heteroaryl used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refers to an optionally substituted group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
  • Non limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4 H -quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl.
  • a heteroaryl group may be mono- or bicyclic.
  • the term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring", “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
  • Heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • nitrogen also includes a substituted nitrogen.
  • Heterocyclic As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable optionally substituted 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more heteroatoms, as defined above.
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • Unsaturated As used herein, the term "unsaturated”, means that a moiety has one or more double or triple bonds.
  • Partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • compounds of the invention may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 ) 0-2 R • , - (haloR • ), -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR • , -(CH 2 ) 0-2 CH(OR • ) 2 ; -O(haloR • ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R • , -(CH 2 ) 0-2 C(O)OH, -(CH 2 ) 0-2 C(O)OR • , -(CH 2 ) 0-2 SR • , -(CH 2 ) 0-2 SH, -(CH 2 ) 0-2 NH 2 , - (CH 2 ) 0-2 NHR • , -(CH 2 ) 0-2 NR • 2 ,
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: -O(CR* 2 ) 2-3 O-, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, -R', -(haloR*), -OH, - OR • , -O(haloR • ), -CN, -C(O)OH, -C(O)OR*, -NH 2 , -NHR • , -NR • 2 , or -NO 2 , wherein each R • is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R ⁇ , -NR ⁇ 2 , -C(O)R ⁇ , -C(O)OR ⁇ , -C(O)C(O)R ⁇ , -C(O)CH 2 C(O)R ⁇ , -S(O) 2 R ⁇ , -S(O) 2 NR ⁇ 2 , - C(S)NR ⁇ 2 , -C(NH)NR ⁇ 2 , or -N(R ⁇ )S(O) 2 R ⁇ ; wherein each R ⁇ is independently hydrogen, C 1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrence
  • Suitable substituents on the aliphatic group of R ⁇ are independently halogen, -R*, -(haloR • ), -OH, -OR • , -O(haloR • ), -CN, -C(O)OH, -C(O)OR • , NH 2 , -NHR • , -NR • 2 , or -NO 2 , wherein each R • is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable protecting group refers to amino protecting groups or hydroxyl protecting groups depending on its location within the compound and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999 .
  • Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di- t -butyl-[9-(10,10-dioxo-10,10,10, 10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-
  • Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t -butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p -methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl ( p -AOM), guaiacolmethyl (GLTM), t -butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-
  • the protecting groups include methylene acetal, ethylidene acetal, 1- t -butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p -methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho
  • a chemical variable e.g., an R group
  • R group on such a ring can be attached at any suitable position, this is generally understood to mean that the group is attached in place of a hydrogen atom on the parent ring. This includes the possibility that two R groups can be attached to the same ring atom.
  • each may be the same or different than other R groups attached thereto, and each group is defined independently of other groups that may be attached elsewhere on the same molecule, even though they may be represented by the same identifier.
  • Antibody includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies) and the term “antibody” is used interchangeably with the terms “immunoglobulin,” “immunoglobulins” and “immunoglobulin molecule”.
  • Each antibody molecule has a unique structure that allows it to bind its specific antigen, but all antibodies have the same overall structure as described herein.
  • the basic immunoglobulin structural unit is known to comprise a tetramer of subunits.
  • Each tetramer has two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • an antibody as defined herein can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2).
  • variable regions and constant regions See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 .
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • FR relatively conserved framework regions
  • CDRs complementarity determining regions
  • Igs immunoglobulins
  • IgA immunoglobulin A
  • IgE immunoglobulin A
  • IgM immunoglobulin M
  • IgD subtypes of IgGs
  • IgG1, IgG2, IgG3 and IgG4 The term is used in the broadest sense and includes single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions which will bind to multiple epitopes or antigens.
  • the terms specifically cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), and antibody fragments so long as they contain or are modified to contain at least the portion of the C H 2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the C H 2 domain, or a variant thereof. Included within the terms are molecules comprising at least the Fab region.
  • antibodies further includes chemical analogues and derivatives of antibodies and antibody fragments, provided that the antibody or antibody fragment maintains its ability to bind specifically to its target antigen.
  • chemical modifications are possible (e.g., glycosylation, acetylation, PEGylation and other modifications without limitation) provided specific binding ability of the antibody is retained.
  • An antibody may be, for example, human, humanized, or chimeric
  • a “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies (mAbs) are highly specific, being directed against a single antigenic site.
  • mAbs monoclonal antibodies
  • each mAb is directed against a single determinant on the antigen.
  • monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
  • the term "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975) Nature, 256:495 , or may be made by recombinant DNA methods ( See , for example, U.S. Pat. No. 4,816,567 to Cabilly et al .).
  • Monoclonal antibodies further include chimeric antibodies in which a portion of the heavy and/or light chain is identical to or homologous with the corresponding s of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • an “intact antibody” is one that comprises an antigen-binding variable region as well as a light chain constant domain (C L ) and heavy chain constant domains, C H 1, C H 2, C H 3 and C H 4, as appropriate for the antibody class.
  • the constant domains may be native sequence constant domains such as human native sequence constant domains or amino acid sequence variants thereof.
  • An intact antibody may or may not have one or more "effector functions", which refers to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • antibody fragment comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragment(s) produced by a Fab expression library, camelids, or an epitope-binding fragments of any of the above which immunospecifically bind to a target antigen (e.g. , a cancer cell antigen).
  • a target antigen e.g. , a cancer cell antigen
  • Humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • the term "capable of specific binding” refers to protein or peptide (e.g., antibody) binding to a predetermined target substance (e.g., an antigen and/or groups of antigens), e.g. a target substance that is expressed on the surface of a cell; thus the term "binding to a target cell” or “binding to a cancer cell” is to be understand as referring to protein or peptide (e.g., antibody) binding to a predetermined target substance (e.g. antigen or antigens) that is expressed on such a cell.
  • a predetermined target substance e.g., an antigen and/or groups of antigens
  • the protein or peptide binds with an affinity of at least about 1 ⁇ 10 7 M 1 , and/or binds to the predetermined target substance (e.g., antigen, antigens or cell) with an affinity that is at least two-fold greater than its affinity for binding to a non-specific control substance (e.g., BSA, casein, non-cancer cells) other than the predetermined target substance or a closely-related target substance.
  • a non-specific control substance e.g., BSA, casein, non-cancer cells
  • drugs refers to small molecules or biomolecules that alter, inhibit, activate, or otherwise affect a biological event.
  • drugs may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, anti-diabetic substances, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, anti-pyretics, steroidal and non-ster
  • polymer As used herein, a "polymer” or “polymeric structure” is a structure that includes a string of covalently bound monomers.
  • a polymer can be made from one type of monomer or more than one type of monomer.
  • the term “polymer” therefore encompasses copolymers, including block-copolymers in which different types of monomer are grouped separately within the overall polymer.
  • a polymer can be linear or branched.
  • treat refers to the administration of a conjugate of the present disclosure to a subject in need thereof with the purpose to alleviate, relieve, alter, ameliorate, improve or affect a condition (e.g., diabetes, cancer, inflammatory disease), a symptom or symptoms of a condition (e.g., hyperglycemia), or the predisposition toward a condition.
  • a condition e.g., diabetes, cancer, inflammatory disease
  • a symptom or symptoms of a condition e.g., hyperglycemia
  • phosphonate-based linkers comprising a monophosphonate, diphosphonate, triphosphonate, or tetraphosphonate group having the general formula
  • R 1 is a first linker arm comprising a tuning element, an optional spacer element, and a reactive functional group capable of reacting with a group on a ligand or targeting moiety in which the tuning element is linked to the O group of the phosphonate group
  • R 2 is a second linker arm comprising a payload linked to the P atom of the phosphonate group via a linker
  • Interspersed between the tuning element and the reactive functional group of the linker arm may be an optional spacer element.
  • the phosphonate-based linker comprises a compound that has the following formula (I) Wherein V is selected from O and S; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, - N(R)C(O)-, -C(O)N(R)-, -S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic
  • the compound may further comprise a cell-specific targeting ligand (L); wherein the compound comprises a compound having formula (II) wherein the reactive functional group (Z) is conjugated to an S, NR, or O group on the cell-specific targeting ligand (L).
  • the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the therapeutic agent.
  • the phosphonate-based linker comprises a compound that has the following formula (IV) Wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, hetero
  • the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload.
  • Such a compound comprises formula (V) Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; or nucleoside;
  • the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload.
  • Such a compound comprises formula (VIII) Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; or nucleoside;
  • the phosphonate-based linker is a compound that has the following formula (X) Wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, hetero
  • the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload.
  • Such a compound comprises formula (XI) Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; or nucleoside;
  • the phosphonate-based linker comprises a compound that has a formula selected from wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroary
  • the phosphonate-based linker comprises a compound that has a formula selected from wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, - S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroary
  • Z may have the structure wherein the wavy line marks the covalent bond between Z and Y, or X when Y is a covalent bond.
  • the linkage Z when conjugated to a cell-specific targeting ligand L, Z may have the structure wherein the wavy lines mark the covalent bond between Z and Y, or X when Y is a covalent bond on the left and Z and L on the right.
  • the O-X linkage of the phosphonate group is stabile extracellularly and labile intracellularly, for example, when present in the lysosomal compartment of the target cell the O-X linkage is cleaved.
  • the phosphonate-W-payload linkage is stable both intracellularly and extracellularly.
  • the payload-W is released with the W attached to a monophosphonate group, which renders the payload charged or polar and facilitates retention of the payload within the cell.
  • n 1, 2, 3, or 4 and the other consituents are as above. It should be noted that even in the case where the conjugate comprises a diphosphonate, triphosphonate, or tetraphosphonate, upon intracellular cleavage, the payload-W is released with the W linked to a monphosphonate.
  • the tuning element provides a tunable stability to the phosphonate linkage when the conjugate is within the lysosomal compartment of the target cell.
  • the intracellular stability of the phosphonate group or rate of intracellular release of the payload from the conjugate may be adjusted or tuned by the particular tuning element adjacent to the phosphonate group and/or by adjusting the number of the phosphonate groups.
  • the conjugates disclosed herein are particularly useful in embodiments in which the ligand is an antibody or antibody fragment and the payload is a therapeutic agent, for example, a cytotoxin or a glucocorticoid receptor agonist, which herein is referred to as an "antibody drug conjugate" or "ADC".
  • the link between the antibody and the drug moiety plays an important role in an antibody drug conjugate (ADC), as the type and structure of the linker may significantly affect the potency, selectivity, and the pharmacokinetics of the resulting conjugate ( Widdeson et al, J. Med. Chem. 49: 4392-4408 (2006 ); Doronina et al., Bioconj. Chem. 17: 114-124 (2006 ); Hamann et al., Bioconj. Chem. 16: 346-353 (2005 ); King et al., J. Med. Chem. 45: 4336-4343 (2002 ); Alley et al., Bioconj.
  • ADC antibody drug conjugate
  • ADC delivery of a drug moiety to its intracellular target occurs via a multistep sequence of events: binding to the cell surface, endocytosis, trafficking (within an endosome) to a lysosome, proteolytic degradation of the conjugate, and diffusion of the released drug moiety across the lysosomal or endosomal membrane toward its intracellular target and its interaction with the target. Therefore, the linker should be sufficiently stable while in circulation to allow delivery of the intact ADC to the target cell but, on the other hand, sufficiently labile to allow release of the drug moiety from the ADC once inside the targeted cell.
  • linkers In general, four types of linkers have been used for preparation of ADCs that have currently entered the clinic: (a) acid-labile linkers, exploiting the acidic endosomal and lysosomal intracellular microenvironment (Hamann et al., op. cit.; Blättler et al., op. cit.); (b) linkers cleavable by lysosomal proteases (Dronina et al. op. cit.; King et al. op. cit.); (c) chemically stable thioether linkers that release a lysyl adduct after proteolytic degradation of the antibody inside the cell; ( Lewis et al Cancer Res.
  • U.S. Patent No.5,094,848 discloses conjugates comprising a diphosphate or amidated diposphate group and a linker arm wherein the linker arm may preferably be an oligopeptide having preferably 2-10 amino acids.
  • the payload-linker conjugates of the present invention wherein the payload is covalently linked to a tuning element of the linker via a monophosphonate, diphosphonate, triphosphonate, or tetraphosphonate linkage have a differentiated and tunable stability of the phosphonate linkage in blood vs. an intracellular environment (e.g. lysosomal compartment).
  • conjugates that have a phosphonate group linking a payload to a tuning element of the linker are stable in circulation (plasma or blood) but reactive in intracellular compartments (e.g., lysosomes) making them suitable for intracellular delivery of payload conjugates where it desired to render the poayload charged to facilitate intracellular retention.
  • the exemplary payload-phosphonate-based linker conjugates in the Examples show that the payload-phosphonate-based linker conjugates of the present invention are stable in blood, which is advantageous for extending the half-life and to prevent premature release of payload from the conjugates but when cleaved intracellularly provides a payload comprising the phosphonate group, which facilitates intracellular retention.
  • the inventors have discovered that by modifying the tuning element and/or V and/or W, and/or the number of phosphonate groups, the ability to tune reactivity or cleavage of the phosphonate linkage in a lysosomal environment so as to release the payload-phosphonate from the conjugate.
  • the rate of release of the payload conjugated to a phosphonate group is dependent on the proximal substitution of the tuning element.
  • the ability to cleave the linkage between the phosphonate and the tuning element efficiently in a lysosome is advantageous for the release of the payload from the conjugate once it has been delivered to a cell and internalized through an endosomal pathway.
  • the excellent solubility of the payload-phosphonate-based linker facilitates conjugation to a ligand or cell-targeting moiety and minimizes aggregation of the conjugates.
  • the phosphonate contributes to retention of the payload to the conjugate within cell and limits permeability of conjugates containing the payload from entering non-target cells.
  • the phosphonate-based linkers provide greater solubility relative to disulfide linkers, cathepsin B-cleavable linkers, esters and acid-sensitive linkers such as hydrazones. They enable the release of the payload conjugated to a phosphonate unlike some of the alternative linkers, and may offer an improved blood/lysosome stability profile.
  • these phosphonate-based linkers will provide superior blood stability relative to esters and disuflides.
  • Phosphonate-based linkers following lysosomal cleavage will release a phosphonate-containing payload.
  • the enzymatic hydrolysis of the phosphonate may be more rapid than the acid-hydrolysis of hyrdazones.
  • the phosphonate-based linkers disclosed herein minimize the propensity for conjugates comprising particular payloads to aggregate.
  • antibody-drug conjugates comprising duocarmycin are known to have a propensity to aggregate.
  • antibodies conjugated to duocarmycin via a phosphonate-based linker disclosed herein did not produce detectable aggregates.
  • the phosphonate-based linkers disclosed herein are particularly useful for conjugating payloads that are prone to forming aggregates to a cell-specific targeting ligand to provide a conjugate with a reduced or no detectable propensity for aggregation.
  • the phosphonate-based linkers disclosed herein provide an ideal design for antibody-drug conjugates and the like.
  • the phosphonate group comprising the phosphonate-based linkers disclosed herein may comprise 1, 2, 3, or 4 phosphate atoms.
  • the phosphonate group may be a phosphonate a diposphonate a triphosphonate tetraphosphonate a phosphorthionate or a diphosphorthionate
  • the wavy lines shown indicate the bond between the P and a linker W connected to a payload (left) and the bond between the O and the tuning element on the proximal end (right).
  • Payloads depicted as "D" herein, are provided in the current invention as part of a payload-ligand conjugate where the payload is a therapeutic agent and is linked to a ligand via a phosphonate-based linker comprising reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadiene, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S.
  • a phosphonate-based linker comprising reactive functional group selected from the group consisting of N-hydroxysuccinimi
  • the payload must possess a desired biological activity and contain a reactive functional group capable of forming a covalent linkage to the linker that attached to the phosphonate group.
  • the desired biological activity includes the diagnosis, cure, mitigation, treatment, or prevention of disease in an animal such as a human.
  • the term "payload” refers to chemicals recognized as drugs in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, or official National Formulary, or any supplement thereof. Exemplary drugs are set forth in the Physician's Desk Reference (PDR) and in the Orange Book maintained by the U.S. Food and Drug Administration (FDA).
  • the functional groups on the drug include primary or secondary amines, hydroxyls, sulfhydryls, carboxyls, aldehydes, and ketones.
  • the drug must have at least one, but may have 2, 3, 4, 5, 6 or more reactive functional groups.
  • the payload may also be a biomolecule such as a peptide, polypeptide, or protein; a nucleic acid molecule or analog thereof, a carbohydrate, polysaccharide, a saccharide, or any other therapeutic agent that has a biological effect.
  • the payload-ligand conjugate is effective for the usual purposes for which the corresponding drugs are effective, but have superior efficacy because of the ability, inherent in the ligand, to transport the drug to the desired cell where it is of particular benefit.
  • exemplary drugs include proteins, peptides, and small molecule drugs containing a functional group for linkage to the phosphonate moiety of the linker.
  • these drugs include, for example, the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, glutocorticoid receptor agonists, nuclear recemptor agonists, antinflammatory agents, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drags, diynenes, the podophyllotoxins, differentiation inducers, and taxols.
  • the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors
  • DNA intercalators glutocorticoid receptor agonists, nuclear recemptor agonists, antinflammatory agents, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the
  • the drugs of the current invention include cytotoxic drugs useful in cancer therapy and other small molecules, proteins or polypeptides with desired biological activity, such as a toxin.
  • the drug may be selected to be activated at a tumor cells by conjugation to a tumor- specific ligand.
  • These tumor specific drug-ligand conjugates have tumor specificity arising from the specificity of the ligand. Examples of this are drug-ligand conjugates that are highly selective substrates for tumor specific enzymes, where these enzymes are present in the proximity of the tumor in sufficient amounts to generate cytotoxic levels of free drug in the vicinity of the tumor.
  • Cytotoxic drugs useful in the current invention include, for example, duocarmycins and CC-1065, and analogues thereof, including CBI (1,2,9,9a- tetrahydrocyclopropa[c]benz[e]indol-4-one)-based analogues, MCBI (7-methoxy- 1,2,9,9a- tetra-hydrocyclopropa[c]benz[e]indol-4-one)-based analogues and CCBI (7-cyano- 1,2,9,9a- tetra-hydrocyclo-propa[c]benz[e]-indol-4-one)-based analogues of the duocarmycins and CC- 1065, doxorubicin and doxorubicin conjugates such as morpholino-doxorubicin and cyanomorpholino-doxorubicin, dolastatins such as dolestatin-10, combretastatin, calicheamicin
  • Anti-inflammatory agents such as glucocorticoid receptor agonists include glucocorticoids such as Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, or mometasone.
  • glucocorticoids such as Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, or mometasone.
  • the phosphonate-based linkers disclosed herein comprise a first arm comprising a tuning element having a distal end and a proximal end wherein the distal end is covalently linked to an oxygen atom of the phosphonate group and the proximal end is covalently linked to a functional reactive group capable of covalent linkage to a cell-targeting ligand and a second arm comprising a linker having a distal end and a proximal end wherein the distal end is covalently linked to a payload or drug and a proximal end covelntly linked to a phophorus atom of the phosphonate group.
  • the linker arm may further include a spacer element interposed between the tuning element and the reactive functional group.
  • tuning elements comprising the first arm include but are not limited to R 1 and R 2 each independently any amino acid,
  • the wavy lines indicate the covalent attachment sites to an oxygen atom of the phosphonate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.
  • tuning elements include but are not limited to The wavy lines indicate the covalent attachment sites to an oxygen atom of the phosphonate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.
  • the spacer element is to allow for distance control away from the cell-targeting ligand. In some embodiments, this distance may have an impact on the stability/cleavability of the linker.
  • the spacer element may be selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, -S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group.
  • the linker comprising the second arm is to allow for distance control away from the phosphonate group. In some embodiments, this distance may have an impact on the efficacy of the payload or drug.
  • the linker may be selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, -S(O)-, -S(O) 2 -, -N(R)SO 2 -, SO 2 N(R)-, a heterocyclic group, an aryl group, or a heteroaryl group.
  • the linker may be a straight polyethylglycol (PEG) chain (of a defined length, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polyethylene groups) and straight carbon chains of C1-30 hysdrocarbons with or without solubilizing groups attached thereto.
  • PEG polyethylglycol
  • the phosphonate linker and payload may be linked to a targeting ligand that selectively delivers a pay load to a cell, organ, or region of the body.
  • exemplary targeting ligands such as antibodies (e.g., chimeric, humanized and human), ligands for receptors, lectins, saccharides, and the like are recognized in the art and are useful without limitation in practicing the present invention.
  • Other targeting ligands include a class of compounds that do not include specific molecular recognition motifs include macromolecules such as poly(ethylene glycol), polysaccharide, polyamino acids and the like, which add molecular mass to the cytotoxin. The additional molecular mass affects the pharmacokinetics of the payload, e.g., serum half-life.
  • the invention provides a payload, linker or payload-linker conjugate with a targeting ligand that is a biomolecule, e.g, an antibody, receptor, peptide, lectin, saccharide, nucleic acid or a combination thereof.
  • a targeting ligand that is a biomolecule, e.g, an antibody, receptor, peptide, lectin, saccharide, nucleic acid or a combination thereof.
  • Biomolecules useful in practicing the present invention may be derived from any source. The biomolecules may be isolated from natural sources or may be produced by synthetic methods. Proteins may be natural proteins or mutated proteins. Mutations may be effected by chemical mutagenesis, site-directed mutagenesis or other means of inducing mutations known to those of skill in the art. Proteins useful in practicing the instant invention include, for example, enzymes, antigens, antibodies and receptors.
  • Antibodies may be either polyclonal or monoclonal, but most preferably are monoclonal and may be human, humanized, or human chimeric antibodies. Peptides and nucleic acids may be isolated from natural sources or can be wholly or partially synthetic in origin.
  • the targeting ligand is an antibody, or antibody fragment, that is selected based on its specificity for an antigen expressed on a target cell, or at a target site, of interest.
  • an antibody or antibody fragment
  • a wide variety of tumor-specific or other disease-specific antigens have been identified and antibodies to those antigens have been used or proposed for use in the treatment of such tumors or other diseases.
  • the antibodies that are known in the art may be used in the conjugates of the invention, in particular for the treatment of the disease with which the target antigen is associated.
  • Targeting ligands may be attached to the linker arm by any available reactive group that can react with the reactive functional group on the proximal end of the linker arm.
  • peptides and proteins may be attached through an amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group may reside at a peptide terminus or at a site internal to the peptide chain.
  • Nucleic acids may be attached through a reactive group on a base (e.g., exocyclic amine) or an available hydroxyl group on a sugar moiety (e.g., 3'- or 5'- hydroxyl).
  • the peptide or protein may be further derivatized at one or more sites to allow for the attachment of appropriate reactive groups onto the peptide or protein. See, Chrisey et al. Nucleic Acids Res. 24:3031-3039 (1996 ).
  • the protein or peptide may be synthesized to contain one or more nonnatural amino acids which may then serve as a site for attachment of the linker arm comprising the payload-phosphonate-based linker.
  • Antibodies comprising nonnatural amino acids for conjugation and methods for making such antibodies have been disclosed in U.S. Patent No. 7,632,924 .
  • the IgG4 constant domain can differ from the native human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 in the KABAT system, wherein the native serine at position 108 (Ser108) of the HC constant domain is replaced with proline (Pro), in order to prevent a potential inter-chain disulfide bond between the cysteine at position 106 (Cys106) and the cysteine at position 109 (Cys109), which correspond to to positions Cys226 and Cys229 in the EU system and positions Cys239 and Cys242 in the KABAT system) that could interfere with proper intra-chain disulfide bond formation.
  • the native human IgG4 constant domain Sewiss-Prot Accession No. P01861.1
  • the antibody may be synthesized to contain one or more non-natural amino acids, the side chain thereof which may then serve as a site for attachment of the linker arm comprising the payload-phosphate-based linker.
  • Antibodies comprising non-natural amino acids for conjugation and methods for making such antibodies have been disclosed in U.S. Patent No. 7,632,924 .
  • the antibody may comprise a substitution of an amino acid residue in the heavy chain or light chain with the non-natural amino acid para-azidophenylalanine (pAzF).
  • the azido group on the side chain of the pAzF residue may be conjugated to a reactive functional group of the therapeutic agent-linker such as a strained cycloalkyne, for example, cyclooctyne.
  • the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSGSSSYLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYGSSPIT; SEQ ID NO: 10) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYAIN; SEQ ID NO: 11), CDR2 (RIIPILDIADYAQKFQD; SEQ ID NO:12), and CDR3 (KDWFDP; SEQ ID NO: 13).
  • the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSFLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO:2), and CDR3 (QQYSSSPLT; SEQ ID NO:3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYPIN; SEQ ID NO: 14), CDR2 (RIIPILGIADYAQRFQG; SEQ ID NO:8), and CDR3 (RDWGDY; SEQ ID NO:9).
  • CDR1 RASQSVSSSFLA
  • CDR2 GASSRAT; SEQ ID NO:2
  • CDR3 QQYSSSPLT
  • CDR3 heavy chain complementarity-determining region
  • the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO: 14.
  • the antibody is a chimeric, humanized, or fully human anti-CD25 antibody that competes with any one of the aforementioned antibodies for binding to the CD25.
  • the aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Patent No. 7,438,907 .
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO:15), CDR2 (YDASNRAT; SEQ ID NO:16), and CDR3 (QQRTNWPLT; SEQ ID NO:17) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SYIMH; SEQ ID NO: 18), CDR2 (VISYDGRNKYYADSVK; SEQ ID NO: 19), and CDR3 (DTDGYDFDY; SEQ ID NO:20).
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSALA; SEQ ID NO:21), CDR2 (DASSLES; SEQ ID NO:22), and CDR3 (QQFNSYPFT; SEQ ID NO:23) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (YYAMH; SEQ ID NO:24), CDR2 (VISYDGSIKYYADSVK; SEQ ID NO:25), and CDR3 (EGPYSNYLDY; SEQ ID NO:26).
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:27), CDR2 (AASSLQS; SEQ ID NO:28), and CDR3 (QQYNSYPLT; SEQ ID NO:29) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DYGMH; SEQ ID NO:30), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:31), and CDR3 (DSIVMVRGDY; SEQ ID NO:32).
  • CDR1 red chain complementarity-determining region
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:33), CDR2 (AASSLQS; SEQ ID NO:34), and CDR3 (QQYNSYPLT; SEQ ID NO:35) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DHGMH; SEQ ID NO:36), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:37), and CDR3 (DSIMVRGDY; SEQ ID NO:38).
  • CDR1 light chain CDR sequences CDR1
  • AASSLQS AASSLQS
  • CDR3 QQYNSYPLT
  • CDR3 heavy chain complementarity-determining region
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO:15), CDR2 (DASNRAT; SEQ ID NO:39), and CDR3 (QQRSNWPLT; SEQ ID NO:40) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SDYYYWS; SEQ ID NO:41), CDR2 (YIYYSGSTNYDPSLKS; SEQ ID NO:42), and CDR3 (GDGDYGGNCFDY; SEQ ID NO:43).
  • CDR1 red chain complementarity-determining region
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.
  • the antibody is a chimeric, humanized, or fully human anti-CD70 antibody that competes with any one of the aforementioned antibodies for binding to the CD70.
  • the aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Patent No. 8,124,738 .
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:44), CDR2 (TVSNRFS; SEQ ID NO:45), and CDR3 (SQSSHVPPT; SEQ ID NO:46) and the heavy chain CDR sequences CDR1 (NYGVN; SEQ ID NO:47), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:48), and CDR3 (SRGKNEAWFAY; SEQ ID NO:49).
  • CDR light chain complementarity-determining region
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) selected from SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49.
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74.
  • Antibodies comprising said CDR sequences have been disclosed in U.S. Patent No. 7,772,373 .
  • the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.
  • HC heavy chain
  • LC light chain
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ASGRYYGSGSYSSYFD; SEQ ID NO:54); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSIK; SEQ ID NO:55), and CDR3 (ARGREYTSQNIVILLD; SEQ ID NO:56); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from AAS, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59.
  • the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74.
  • Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No.
  • the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.
  • HC heavy chain
  • LC light chain
  • the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising the light chain CDR sequences CDR1 (ASQSVSSDV; SEQ ID NO:60), CDR2 (YAS), and CDR3 (QDYTSPRT; SEQ ID NO:61) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63); or the light chain CDR sequences CDR1 (ASQSVSHDV; SEQ ID NO:54), CDR2 (YTS), and CDR3 (QDYSSPRT; SEQ ID NO:65) and the heavy chain CDR sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63).
  • ASQSVSSDV the light chain CDR sequences CDR1
  • the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from YYAS, YSG, YTS, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65.
  • the antibody is a chimeric, humanized, or fully human anti-CD163 antibody that competes with any one of the aforementioned antibodies for binding to the CD163.
  • Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No. 20120258107 and 20120276193 .
  • the antibody has reduced effector function or lacks effector function compared to a wild-type or native IgG1 antibody. Reducing or eliminating effector function may be achieved by providing an antibody with an IgG4 framework or constant domain.
  • the IgG4 constant domain may differ from the native human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 as determined in the KABAT numbering scheme (See, e.g., Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed.
  • the antibody that has reduced or lacks effector function is an aglycosylated antibody that lacks the N-glycan at position 297 of the heavy chain (as determined using the KABAT Numbering scheme).
  • Aglycosylated antibodies may be produced in a prokaryote expression system, for example, E. coli.
  • the antibody may be encoded by a nucleic acid molecule that introduces an amino acid substitution in any of positions 297-299 of the heavy chain such that the antibody is substantially aglycosylated when the nucleic acid molecule is expressed in a mammalian cell.
  • the glycosylation site is Asn297 within the amino acid sequence QYNS (SEQ ID NO:66).
  • the glycosylation site corresponds to Asn297 of IgG1.
  • the glycosylation site is the asparagine within the amino acid sequence QFNS (SEQ ID NO:67). Accordingly, a mutation of Asn297 of IgG1 removes the glycosylation site in an Fc portion derived from IgG1. In one embodiment, Asn297 is replaced with Gln.
  • the tyrosine within the amino acid sequence QYNS (SEQ ID NO:66) is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation.
  • a T-cell epitope is a polypeptide sequence in a protein that interacts with or binds an MHC class II molecule.
  • the amino acid sequence QYNS (SEQ ID NO:66) within an IgG1 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence.
  • a mutation of asparagine within the amino acid sequence QFNS (SEQ ID NO:67) removes the glycosylation site in an Fc portion derived from IgG2 or IgG4 heavy chain.
  • the asparagine is replaced with a glutamine.
  • the phenylalanine within the amino acid sequence QFNS is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation.
  • the amino acid sequence QFNS (SEQ ID NO:67) within an IgG2 or IgG4 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence.
  • the antibody comprises a substitution of one or more of the amino acids at position 318, 320, 322, 234, 235, 236, 237, or 297 of the antibody wherein the antibody with the substitution has a reduced effector function compared to an antibody comprising the native or wild-type amino acid at the position.
  • the effector function may be binding affinity for Clq and/or binding affinity for the Fc receptor.
  • the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264.
  • the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine.
  • the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
  • a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier.
  • the compounds described herein including pharmaceutically acceptable carriers such as addition salts or hydrates thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.
  • the conjugates of the invention comprising an antibody or antibody fragment as the targeting moiety may be administered parenterally, more preferably intravenously.
  • administering or “administration” are intended to encompass all means for directly and indirectly delivering a compound to its intended site of action.
  • the compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof may be administered singly, in combination with other compounds of the invention, and/or in cocktails combined with other therapeutic agents.
  • the choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxyniethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injection is a preferred method of administration for the compositions of the current invention.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection or a transdermal patch.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Step A Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-17-(non-8-enoyloxy)-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid (16)
  • Step B Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl non-8-enoate ( 17 )
  • Step C Preparation of (9-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-9-oxononyl)phosphonic acid ( 18 )
  • This example shows the synthesis of intermediate 25 for constructing A-Ring Analogs.
  • Step A Preparation of 4-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-13b-(2-((tertbutyldimethylsilyl)oxy)acetyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4",5":3',4']cyclopenta[1',2':5,6]naphtho[1,2-f]indazol-8(3aH,4bH,13bH)-yl)phenyl pivalate (22)
  • Step B Preparation of 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(4-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1.3]dioxolo[4",5":3',4']cyclopenta[1',2':5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (intermediate 23)
  • This example shows the synthesis of A-Ring Analogs.
  • Step A Preparation of 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-8-(3-(hex-5-en-1-yloxy)phenyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5, 6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4",5":3';4']cyclopenta[1',2':5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (26)
  • Step B Preparation of (6-(3-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-13b-(2-hydroxyacetyl)-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1.3]dioxolo[4",5":3',4']cyclopenta[1',2':5,6]naphtho[1,2-f]indazol-8(3aH,4bH,13bH)-yl)phenoxy)hexyl)phosphonic acid (27)
  • This example shows the synthesis of pyrophosphonate drug linkers.
  • Step B Preparation of ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio) decyl) phosphonic anhydride ( 34 )
  • Step C Preparation of (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S, 16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9, 10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio) decyl) phosphonic anhydride ( 35 )
  • the resulting solution was stirred at ambient temperature for 1h and determined to be complete as judged by LCMS.
  • the reaction was concentrated and taken up in methanol and injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini -NX C18 OBD 5 uM 30 x 100mm; 10-70%MeCN/water w/ 0.1% NHaOH modifier over 20 min, 235nM wavelength).
  • Step D Preparation (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl)phosphonic anhydride (37)
  • (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S, 16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-hydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl)phosphonic anhydride ( 35 , 9.0mg, 11mmol, 1.0 eq) and 2-(cyclooct-2-yn-1-yloxy)acetic acid ( 36 , 3.0 mg, 17 mmol, 1.5eq) were dissolved in anhydrous DMF (0.37mL, 0.03M).
  • This example shows the synthesis of pyrophosphonate drug linkers.
  • Step A 1,3-dioxoisoindolin-2-yl 2-(cyclooct-2-yn-1-yloxy)acetate(38)
  • Step B (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphonic acid (40)
  • Step C (2-(2-(cyclooct-2yn-1-yloxy)acetamido)ethyl phosphoric) (6-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((ftuoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-6-oxohexyl)phosphonic anhydride (41)
  • Step A Preparation of 2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (46)
  • Step B Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17(((10-(((4-(2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)decyl)thio)carbonyl)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate ( 47 )
  • This example shows the synthesis of various antibody drug conjugates (ADCs).
  • ADCs antibody drug conjugates
  • Site specific conjugation using click (2 + 3) chemistry Para-azido phenylalanine containing anti-CD74 antibodies were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; 0-20% Dimethylamine, pH 6.0 and concentrated to 1-20mg/mL. 10-15 molar equivalents of cyclooctyne drug-linker were added and reacted for 16- 72 hours at 28-30°C.
  • the antibody conjugates were purified over a SP 650S column (Tosoh Biosciences) to remove excess reagents.
  • conjugates were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0, 0.22 ⁇ m filtered, and stored at 4°C.
  • ADC number Structure ⁇ CD74-47 ⁇ CD74-50 ⁇ CD74-41
  • the potency of binding to glucocorticoid receptor by small molecule compounds was measured with the PolarScreen TM Glucocorticoid Receptor Competitor Assay Kit, Red (Life Technology, Catalog #A15898) according to the procedure in the kit.
  • the fluormone GS red and GR full length protein were subsequently added into the assay plates, along with the positive and negative controls.
  • the samples were mixed on a shaker for 1 minute and then the plates were incubated at room temperature for 2-4 hours with minimal light exposure.
  • the plates were read with an Envision and the relative fluorescent signals were calculated.
  • the data were plotted in GraphPad Prism and the EC50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism.
  • GILZ gene assays were conducted as follows. HUT78 cells were cultured in IMEM plus 20% heat inactivated FBS and cell density was maintained between 0.1 to 1.2 million / mL. 786-O cells were cultured in RPMI plus 10% heat inactivated FBS. Actively growing cells were harvested and resuspended in HBSS with 2% FBS at 1.1 million cells per mL then dispensed to 384-well V-bottom plates at 45 ⁇ L per well. Serially diluted ADC solution was added to the cell plate (5 ⁇ L per well) and mixed for 2 min. Cells were then cultured at 37 °C, at 5% CO 2 for a designated time before supernatant was removed.
  • Cells were harvested in lysis buffer from the Cells-to-Ct kit (40 ⁇ L, Life Technologies, 4391851C) following the supplier's protocol and mixed for 10 min followed by addition of 5 ⁇ L per well of stop solution from the kit.
  • cDNA was synthesized with a reverse transcription kit (Life Technologies, 4391852C) follow by qPCR using the TaqMan gene expression master mix (Life Technologies, 4369016) with GILZ gene assay (Life Technologies, Hs00608272_m1) and GAPDH assay (Hs2758991_g1) in a duplex format with 3-4 technical replicates.
  • MDCKII cells (kindly provided by the Netherlands Cancer Institute, under a licensing agreement) were seeded on to 96-well transwell culture plates (Millipore Corp, Billerica, MA) and used in experiments after five days in culture.
  • Test compound (1 ⁇ M) was prepared in Hank's Balanced Salt Solution (HBSS), 10 mM (4-(2-hydroxyethyl)-1-piperrazineethanesulfonic acid) (HEPES, pH 7.4), with 10 ⁇ M cyclosporine A (to inhibit endogenous transport) and 1.2 ⁇ M dextran Texas red (to confirm monolayer integrity).
  • Substrate solution 150 ⁇ L was added to either the apical (A) or the basolateral (B) compartment of the culture plate, and buffer (150 ⁇ L; HBSS, 10 mM HEPES, pH 7.4) with 10 ⁇ M cyclosporine A was added to the compartment opposite to that containing the substrate.
  • buffer 150 ⁇ L; HBSS, 10 mM HEPES, pH 7.4
  • 10 ⁇ M cyclosporine A was added to the compartment opposite to that containing the substrate.
  • 50 ⁇ L samples were removed from both sides of monolayers dosed with test compound and placed in 96 well plates, 50 ⁇ L internal standard (1 ⁇ M labetolol) and 100 ⁇ L HBSS was added to the samples.
  • Samples were analyzed by LC/MS/MS using an Applied Biosystems SCIEX API 5000 triple quadruple mass spectrometer (Concord, ON, Canada) with a TurboIonSpray ion source in the positive ion mode.
  • a Thermo Scientific Transcend LX-2 system (Franklin, MA.) was coupled to the API 5000 with a flow rate of 800 ⁇ L/min to direct sample into the mass spectrometer.
  • Volume of Receiver Chamber is 0.15 mL
  • Area of membrane is 0.11 cm 2
  • ⁇ in concentration concentration in the receiver compartment at 3 hr
  • P app was expressed as 10 -6 cm/s.
  • UV The concentration of conjugates was determined by absorbance at 280 nm with a background correction of 320nm using Agilent U.V spectrometer (Model#8453).
  • SEC The ADCs were analyzed by SEC-HPLC (G3000SWXI, Tosoh Column 7.8x300 mm (Serial#Y02322), Mobile Phase -200 mM KPO4, 250 mM KCl, pH 6.0 + 10% IPA; at flow rate 0.5ml/min, isocratic gradient for 40 min, 10 ⁇ g load) on an Agilent 1100 HPLC. The data was processed using the software Agilent Chemstation and the percent monomer was reported.
  • MS and DAR The sample was denatured with 3M guanidine HCl, reduced with 150mM DTT and analyzed by LC-MS (Agilent PLRP-S,4000°A, 8um, 2.1x150mm (Serial#0001023345-118) Mobile Phase A-0.05%TFA in Water and Mobile Phase B- 0.05%TFA in Acetonitrile, at flow rate 0.3ml/min, 80°C for 23 min, 3 ⁇ g load) on Agilent Q-TOF LC/MS (Model#6510). Data was acquired and deconvoluted to monoisotopic and singly charged species using the software Agilent Mass Hunter Qualitative Analysis. The identity was confirmed from the mass of the light chain, the unconjugated and conjugated heavy chains. DAR was calculated from the DAD signal.
  • Residual Drug Linker was extracted from Antibody Drug Conjugate by precipitating out the antibody with ACN:MeOH (50:50). The supernatant was analyzed by RP-HPLC (Waters X Bridge BEH C18 2.1x150mm (PN 186003110), Mobile Phase A - 4%NH4OH, 1.5%FA in water and Mobile Phase B - Acetonitrile; flow rate 0.3ml/min, 60°C for 28 min, 10 ⁇ l load) on Agilent Q TOF LC-MS (Model#6510). The data was processed using Agilent Mass Hunter Qualitative Analysis.
  • the carrier solution for all ADCs and naked antibodies was 50 mM histidine pH 6.0, 100 mM NaCl and 5% trehelose. To assure consistency across experiments, ADCs were thawed on ice, dispensed to small aliquots, frozen to -80 °C, and those aliquots were then used for experiments with unused material discarded at the end of the day. For all in vitro studies in this report, none of the read outs were measurably different in the presence or absence of the carrier solution. Nevertheless, the final concentration of antibody carrier solution was maintained constant at 1 % v/v by serially diluting antibody solution prior to addition to the assay mixture and adding carrier solution to control wells. The same practice was observed for DMSO as the solvent for small molecules, with the final concentration being 0.1% v/v.
  • the ⁇ Ct method was used to calculate fold change of expression of ZBTB16 relative to control sample, using 3 replicates per treatment condition.
  • B cells were seeded at 100,000 cells/well in 90 ⁇ L in a 96-well flat bottom plate. Three fold serial dilutions were performed in DMSO, subsequent intermediate 10x stocks were made in tissue-culture medium and then added to the cell plate (10 ⁇ L/well). Cells were incubated at 37 °C, 5% CO 2 for 18 hr, harvested and processed for PCR as described above.
  • EC50 curves were calculated using Graph Pad Prism 6 software using nonlinear fit (agonist) vs. response - variable slope (four parameters) calculation.

Claims (15)

  1. Composé comprenant la formule :
    Figure imgb0088
     dans laquelle
    V est choisi parmi O et S ;
    W est choisi parmi une chaîne hydrocarbure en C1-30 facultativement substituée, bivalente, linéaire ou ramifiée, saturée ou insaturée, un ou plusieurs motifs méthylène étant facultativement et indépendamment remplacés par -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, - N(R)C(O)-, -C(O)N(R)-, -S(O), -S(O)2-, -N(R)SO2-, SO2N(R)-, un groupe hétérocyclique, un groupe aryle ou un groupe hétéroaryle ;
    X est choisi parmi une liaison covalente ; un atome de carbone ; un hétéroatome ; un groupe facultativement substitué choisi dans le groupe constitué par un acyle, un aliphatique, un hétéroaliphatique, un aryle, un hétéroaryle et un hétérocyclique ; ou un nucléoside ;
    Y est choisi parmi une liaison covalente ou une chaîne hydrocarbure en C1-30 facultativement substituée, bivalente, linéaire ou ramifiée, saturée ou insaturée, un ou plusieurs motifs méthylène étant facultativement et indépendamment remplacés par -O-, -S-, -N(R)-, -C(O)-, C(O)O-, OC(O)-, -N(R)C(O)-, -C(O)N(R)-, -S(O), -S(O)2-, -N(R)SO2-, SO2N(R)-, un groupe hétérocyclique, un groupe aryle ou un groupe hétéroaryle ;
    Z est un groupe fonctionnel réactif choisi dans le groupe constitué par l'ester de N-hydroxysuccinimidyle, le carbonate de para-nitrophényle, le carbamate de para-nitrophényle, l'azoture, l'hydrazine, le pentafluorophényle, l'haloacétamide, le maléimide, l'hydroxylamine, le cycloalcyne filtré et l'hétérocycloalcyne, l'alcyne, le diène, l'azadiène et l'azadiène hétérocyclique, dans lequel le groupe halo est un iode (I), un brome (Br), un fluor (F) ou un chlore (Cl) et le groupe hétéro est N, O ou S ;
    D est un agent thérapeutique ;
    chaque occurrence de R est indépendamment un hydrogène, un groupe de protection approprié, une fraction acyle, une fraction arylalkyle, une fraction aliphatique, une fraction aryle, une fraction hétéroaryle ou une fraction hétéroaliphatique ; et
    n est 1, 2, 3 ou 4.
  2. Composé selon la revendication 1, dans lequel l'agent thérapeutique est un agent cytotoxique, un agent anti-inflammatoire, un peptide, un acide nucléique ou un analogue d'acide nucléique.
  3. Composé selon la revendication 2, dans lequel l'agent anti-inflammatoire est un agoniste du récepteur des glucocorticoïdes.
  4. Composé selon la revendication 2, dans lequel l'agent anti-inflammatoire est le cortisol, l'acétate de cortisone, la béclométasone, la prednisone, la prednisolone, la méthylprednisolone, la bétaméthasone, la triamcinolone, le budésonide, la dexaméthasone, la fluticasone ou la mométasone.
  5. Composé selon la revendication 1 comprenant en outre un ligand de ciblage spécifique des cellules (L) ; dans lequel le composé comprend la formule
    Figure imgb0089
     dans laquelle le groupe fonctionnel réactif (Z) est conjugué à un groupe S, NR ou O sur le ligand de ciblage spécifique des cellules (L).
  6. Composé selon la revendication 5, dans lequel la charge utile est un agent thérapeutique, un marqueur détectable, un radionucléide ou un groupe de protection.
  7. Composé selon la revendication 5, dans lequel l'agent thérapeutique est un agent cytotoxique, un agent anti-inflammatoire, un peptide, un acide nucléique ou un analogue d'acide nucléique.
  8. Composé selon la revendication 7, dans lequel l'agent anti-inflammatoire est un agoniste du récepteur des glucocorticoïdes.
  9. Composé selon la revendication 7, dans lequel l'agent anti-inflammatoire est le cortisol, l'acétate de cortisone, la béclométasone, la prednisone, la prednisolone, la méthylprednisolone, la bétaméthasone, la triamcinolone, le budésonide, la dexaméthasone, la fluticasone ou la mométasone.
  10. Composé selon la revendication 5, dans lequel le ligand de ciblage spécifique des cellules est un anticorps ou un anticorps monoclonal (p. ex. chimérique, humanisé ou humain), un ligand pour un récepteur, une lectine, un saccharide, le poly(éthylène glycol), un polysaccharide ou un acide polyamino.
  11. Composé selon la revendication 5, dans lequel le ligand de ciblage spécifique des cellules est un anticorps anti-Her2, un anticorps anti-CD4, un anticorps anti-CD20, un anticorps anti-EGFR, un anticorps anti-CD22, un anticorps anti-CD23, un anticorps anti-CD25, un anticorps anti-CD52, un anticorps anti-CD30, un anticorps anti-CD33, un anticorps anti-CD40L, un anticorps anti-CD70, un anticorps anti-CD74, un anticorps anti-CD80, un anticorps anti-CD163, un anticorps anti-Mucl8, un anticorps anti-intégrine, un anticorps anti-PSMA, un anticorps anti-CEA, un anticorps anti-CDl Ia, un anticorps anti-CTLA4 ou un anticorps anti-BLys chimériques, humanisés ou humains.
  12. Composé selon l'une quelconque des revendications 1 à 11 pour utilisation dans le traitement d'une maladie ou d'un trouble.
  13. Composé selon la revendication 7, dans lequel l'agent thérapeutique est un agent anti-inflammatoire.
  14. Composé selon la revendication 13, dans lequel l'agent anti-inflammatoire est un agoniste du récepteur des glucocorticoïdes.
  15. Composé selon la revendication 7, dans lequel l'agent thérapeutique est un agent cytotoxi que.
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US20190030171A1 (en) 2019-01-31
US10869929B2 (en) 2020-12-22
US20230147502A1 (en) 2023-05-11
WO2017132103A2 (fr) 2017-08-03
US20210015930A1 (en) 2021-01-21
US11883500B2 (en) 2024-01-30
EP3407916A2 (fr) 2018-12-05
EP3407916A4 (fr) 2019-09-11
US11554172B2 (en) 2023-01-17
WO2017132103A3 (fr) 2018-06-07

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